1,1 &#39;-[[(substituted alkyl)imino]bis(alkylene)]bis-ferrocenes and their use in i electrochemical assays by labelling substrates of interest

ABSTRACT

Compounds of general formula I wherein Fc and Fc′ may be the same or different and are substituted ferrocenyl moieties having at least one ring substituent selected from sulfur-containing groups, phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl groups containing two or more fluorine atoms, heteroaryl, substituted phenyl, and cyano, wherein if present as sole substituent the cyano group is located on the proximal cyclopentadienyl ring; X is a spacer, Y is a spacer, Z is a spacer; and R is a linker group. Compound I may be used to make labelled substrates, functionalised compounds for making labelled substrates and may be used as labels in an electrochemical assay.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/410,296 entitled “1,1′-[[(SUBSTITUTEDALKYL)IMINO]BIS(ALKYLENE)]BIS-FERROCENES AND THEIR USE IN IELECTROCHEMICAL ASSAYS BY LABELLING SUBSTRATES OF INTEREST,” filed onDec. 22, 2014, which is a national stage application, filed under 35U.S.C. § 371, of International Application No. PCT/GB2013/051643, filedon Jun. 21, 2013, and claims the benefit of, and priority to, GB PatentApplication No. 1211157.1, filed on Jun. 22, 2012, the complete contentsof which are hereby incorporated herein by reference in their entiretyfor all purposes.

FIELD OF THE INVENTION

The invention relates to electrochemical detection methods. Moreespecially, the invention relates to electrochemical assays, toelectrochemically active labels for use in electrochemical detectionmethods, and to their use.

BACKGROUND OF THE INVENTION

The detection of certain biological molecules plays an important part inmany aspects of life. For example, in the medical field, there is anever-present need to detect bacterial or viral pathogens, or biologicalmolecules. Other fields in which sensitive assays are essential includethe food and beverage industries.

WO03/074731 discloses a method of probing for a nucleic acid. A nucleicacid solution is contacted with an oligonucleotide probe with anelectrochemically active marker. The probe is caused to at leastpartially hybridise with any complementary target sequence which may bepresent in the nucleic acid solution. Following enzymatic degradation ofthe nucleic acid probe, information is electrochemically determinedrelating to the marker. Compounds for use in the method are alsodisclosed.

WO2005/005657 discloses a method of detecting protease activity in whicha sample solution is contacted with a protease substrate with anelectrochemically active marker, providing conditions under which anyprotease which may be present in the sample may degrade the proteasesubstrate and information relating to the electrochemically activemarker is electrochemically determined. Certain novel compounds for usein the process were also disclosed.

WO2012/085591 describes certain diferrocenyl compounds for use aselectrochemical labels.

There is a continuing need to develop labels that enable detection ofthe presence in small concentrations of biological substrates orindicators, for example, nucleic acids (in isolated form or in the formof larger molecules, for example, natural or syntheticoligonucleotides), or amino acids (in isolated form or in the form oflarger molecules, for example, natural or synthetic peptides). Inparticular, there is a continuing need for new labels with differentoxidation potentials and/or with different chemical or physicalproperties thereby widening the range of possible assays available andincreasing the scope for the development of multiplex reactions.

SUMMARY OF THE INVENTION

The invention provides a compound according to general formula I

in which:

-   -   Fc is a substituted ferrocenyl moiety having at least one ring        substituent selected from sulfur-containing groups,        phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl        groups containing two or more fluorine atoms, heteroaryl,        substituted phenyl, and cyano, wherein if present as sole        substituent the cyano group is located on the proximal        cyclopentadienyl ring;    -   Fc′ is a substituted ferrocenyl moiety having at least one ring        substituent selected from sulfur-containing groups,        phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl        groups containing two or more fluorine atoms, heteroaryl,        substituted phenyl, and cyano, wherein if present as sole        substituent the cyano group is located on the proximal        cyclopentadienyl ring, and may be the same as or different from        Fc;    -   X is a spacer    -   Y is a spacer    -   Z is a spacer; and    -   R is a linker group.

The invention also provides use as a label in an electrochemical assayof a compound of general formula I:

in which:

-   -   Fc is a substituted ferrocenyl moiety, having at least one ring        substituent selected from sulphur-containing groups,        phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl        groups containing two or more fluorine atoms, heteroaryl,        substituted phenyl, and cyano, wherein if present as sole        substituent the cyano group is located on the proximal        cyclopentadienyl ring;    -   Fc′ is a substituted ferrocenyl moiety having at least one ring        substituent selected from sulphur-containing groups,        phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl        groups containing two or more fluorine atoms, heteroaryl,        substituted phenyl, and cyano, wherein if present as sole        substituent the cyano group is located on the proximal        cyclopentadienyl ring, and may be the same as or different from        Fc;    -   X is a spacer    -   Y is a spacer    -   Z is a spacer; and    -   R is a linker group.

The compounds used in accordance with the invention have been found tobe effective labels for use in electrochemical assays. In particular,the compounds may be used to form labelled substrates. Molecules ofinterest as substrates that may be labelled include, but are not limitedto: amino acids, nucleotides, nucleosides, sugars, peptides, proteins,oligonucleotides, polynucleotides, carbohydrates and derivatives orsynthetic analogs of any of those molecules. Other substrates that mightbe labelled using the compounds of the invention includelatex/paramagnetic microparticles and nanoparticles. The labellingcompounds of general formula I and labelled molecules including labelsderivable from the labelling compounds are potentially useful inelectrochemical techniques in which their electrochemicalcharacteristics can be utilized to derive information about the labelsor their environment. For example, the compounds of the invention mayfind use in a method as described in WO03/074731 or in a method asdescribed in WO2005/005657. The labelling compounds of the invention andthe labelled substrates derived therefrom offer characteristics whichmake them useful complements to previously known labelling compounds,permitting a wider spectrum of applications, for example, offeringadditional opportunities for avoidance of conditions under whichmeasurement potential may be compromised by interference with impuritiesthat may be present and/or offering differing electrochemical potentialvalues and/or allowing more greater flexibility in multiplex assays. Anumber of the compounds and the corresponding labelled substrates haverelatively high electro potential values, as illustrated in particularby Example 4 below. It is believed that, especially, compounds of theinvention having sulfur-containing or phosphorus-containing substituentsand their corresponding labelled substrates will be useful in providingfor assays in which the measurement potential will be relatively high,for example, in excess of 400 mV, for example in excess of 450 mV oreven in excess of 500 mV. Compounds having electrochemical potentials ofat least 450 mV, for example 500 mV or more, will be particularly usefulin extending the range of available potential values and therefore, forexample, in potentially providing for more effective multiplex assays.Other compounds of the invention having highly electron-withdrawingsubstituents, for example, trifluoromethyl or cyano, are believed tohave similar advantages in terms of offering high electrochemicalpotential values thereby extending the range of useful labels andlabelled substrates. Additionally, some compounds of the invention andthe corresponding labelled substrates offer the advantage of having anarrower voltage peak, which is advantageous in providing for the optionof utilising a greater number of labels in a multiplex assay, since thenarrower measurement peaks result in wider gaps between peaks, which maybe utilised if desired by incorporating additional labels withpotentials that will be within the gaps.

In all aspects of the invention, the following spacers are preferred: Xis a C1 to C6 alkylene chain which is optionally interrupted by —O—,—S—, or —NR⁵—, in which R⁵ represents hydrogen or C1 to C6 alkyl; Y is aC1 to C6 alkylene chain which is optionally interrupted by —O—, —S—, or—NR⁵—, in which R⁵ represents hydrogen or C1 to C6 alkyl; and Z is a C1to C12 alkylene chain which may optionally be substituted and/or mayoptionally be interrupted by —O—, —S—, cycloalkyl, —CO—, —CONR¹—,—NR¹CO— or —NR¹— in which R¹ represents hydrogen or C1 to C4 alkyl.

In the compounds used according to the invention it is preferred that Xrepresents C1- to C6-alkylene optionally interrupted by oxygen; Yrepresents C1 to C6-alkylene optionally interrupted by oxygen; and Zrepresents C1 to C8 alkylene optionally interrupted by oxygen. Thus inthese embodiments X, Y and Z can be represented by the formula(CH₂)_(a)—O—(CH₂)_(b) wherein a≥0 and b≥0. For X and Y, a+b=1-6. For Z,a+b=1-8. Ideally a≥1 and b≥1.

X is preferably —(CH₂)_(x)— in which x is from 1 to 6, preferably 1 to4, especially 1 or 2; or C1 to C6-alkylene interrupted by oxygen, forexample —(CH₂)₃— O—CH₂—, —(CH₂)₂— O—(CH₂)₂—, or —CH₂— O—(CH₂)₃—.

Y is preferably —(CH₂)_(y)— in which y is from 1 to 6, preferably 1 to4, especially 1 or 2; or C1 to C6-alkylene interrupted by oxygen, forexample —(CH₂)₃— O—CH₂—, —(CH₂)₂— O—(CH₂)₂— or —CH₂— O—(CH₂)₃—.

Preferably X and Y are the same. Preferably Fc and Fc′ are the same andX and Y are the same.

In an embodiment, Z is a C1 to C12 alkylene chain which may optionallybe substituted and/or may optionally be interrupted by —O—, —S— or —NR¹—in which R¹ represents hydrogen or C1 to C4 alkyl. Preferably Z is—(CH₂)_(z)— in which z is from 1 to 8, with z preferably representingfrom 1 to 6, especially from 2 to 6; or is C1 to C8 alkylene interruptedby oxygen, for example —(CH₂)₂—O—(CH₂)₃— or —(CH₂)₃—O—(CH₂)₂—. In onepreferred embodiment: X is —(CH₂)_(x)— in which x is 1 or 2; Y is—(CH₂)— in which y is 1 or 2; and Z is —(CH₂)_(z)— in which z is from 1to 8. Where X and Y represent an alkylene chain interrupted by —NR⁵—, R⁵preferably represents hydrogen or C1 to C4 alkyl, more preferablyhydrogen.

In one preferred embodiment, the invention provides use, as anelectrochemical label, of a compound of the general formula II:

in which:

-   -   Fc is a substituted ferrocenyl moiety as defined above with        reference to general formula I,    -   Fc′ is a substituted ferrocenyl moiety as defined above with        reference to general formula I, and may be the same as or        different from Fc;    -   x is 1 or 2;    -   y is 1 or 2;    -   z is from 1 to 8;    -   and R is a linker group.

Preferably, x and y are each equal to 1.

It is preferred that the ferrocenyl moieties are the same, and it istherefore preferred that Fc and Fc′ carry the same substituents in thesame positions.

Except where the contrary is apparent from the context, the term“substrate” is used throughout the remainder of this document to includeboth naturally occurring substrates and synthetic substrates, andreferences herein to amino acids, nucleotides, nucleosides, sugars,peptides, proteins, oligonucleotides, polynucleotides, or carbohydrates,are to be understood as referring to naturally occurring or syntheticamino acids, nucleotides, nucleosides, sugars, peptides, proteins,oligonucleotides, polynucleotides, or carbohydrates. Substrates can alsobe polypeptides. Synthetic substrates include synthetic analogues ofnaturally occurring substrates. Substrates include single nucleotidesand single amino acids. In the case of an assay relying upon cleavage ofa substrate, for example by an enzyme, a single amino acid may beregarded as a substrate because, although it lacks an internal bondcapable of being cleaved by a protease enzyme, such a bond may be formedthrough the attachment of a marker. Where derivatives of naturallyoccurring substrates are referred to herein, those derivatives may benaturally occurring derivatives or synthetic derivatives of thesubstrate.

The invention provides a method of detecting a chemical entity using acompound according to the invention. Use in an electrochemical assayaccording to the invention may be for example in an assay for detectingan electrochemically labelled substrate. The electrochemical assay mayfor example be an assay for determination of the amount of anelectrochemically labelled substrate. The assay may advantageously befor detecting or determining the amount of a labelled substrate whereinthe labelled substrate is selected from amino acids, nucleotides,nucleosides, sugars, peptides, proteins, oligonucleotides,polynucleotides, carbohydrates, microparticles and nanoparticles. Incertain preferred embodiments, the assay is for detecting or determiningthe amount of a labelled substrate in which the labelled substrate isselected from nucleotides, nucleosides, oligonucleotides, andpolynucleotides. In another advantageous embodiment, the assay is fordetecting or determining the amount of a labelled substrate in which thelabelled substrate is selected from amino acids, peptides, and proteins.

For the purpose of attachment to substrates, the label may befunctionalised by addition of a functionalising group. Thus, theinvention further provides functionalised derivatives comprising amoiety derivable from the compounds of the invention attached to afunctionalising group suitable for enhancing attachment to a substrate.

The invention also provides a method for manufacturing a functionalizedlabelling compound comprising a label moiety for use in anelectrochemical assay, comprising reacting a compound of general formulaI:

in which:

-   -   Fc is a substituted ferrocenyl moiety having at least one ring        substituent selected from sulphur-containing groups,        phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl        groups containing two or more fluorine atoms, heteroaryl,        substituted phenyl, and cyano, wherein if present as sole        substituent the cyano group is located on the proximal        cyclopentadienyl ring,    -   Fc′ is a substituted ferrocenyl moiety having at least one ring        substituent selected from sulphur-containing groups,        phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl        groups containing two or more fluorine atoms, heteroaryl,        substituted phenyl, and cyano, wherein if present as sole        substituent the cyano group is located on the proximal        cyclopentadienyl ring, and may be the same as or different from        Fc;    -   X is a spacer    -   Y is a spacer    -   Z is a spacer; and    -   R is a linker group,

with a functionalising compound to obtain a funtionalised labellingcompound of general formula III:

A-L-F  III

in which A represents

wherein

-   -   Fc, Fc′, X, Y and Z are as defined above with reference to        general formula I;    -   F represents a functionalising moiety, especially a        functionalising moiety for reacting with a substrate for        attachment of the labelling moiety to the substrate; and    -   L represents a linker moiety.

The linker moiety L will generally be a linker moiety derivable from thelinker group R. For example where R is or contains an OH group L willusually represent or comprise —O—.

Furthermore the invention provides a method for the manufacture of alabelled substrate, comprising reacting a compound of general formulaIII:

A-L-F  III

in which A, L and F are as defined above;

with a substrate to form a labelled substrate.

The invention moreover provides a functionalised labelling compound foruse in the manufacture of a labelled substrate, the functionalisedlabelling compound having the general formula III:

A-L-F  III

in which A, L and F are as defined above.

The invention also provides a labelled substrate for use in anelectrochemical assay, the labelled substrate being of general formulaIIIa:

A-L-F′—[S]  IIIa

in which A represents

in which:

-   -   Fc is a substituted ferrocenyl moiety having at least one ring        substituent selected from sulphur-containing groups,        phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl        groups containing two or more fluorine atoms, heteroaryl,        substituted phenyl, and cyano, wherein if present as sole        substituent the cyano group is located on the proximal        cyclopentadienyl ring,    -   Fc′ is a substituted ferrocenyl moiety having at least one ring        substituent selected from sulphur-containing groups,        phosphorus-containing groups, iodo, chloro, silyl, fluoroalkyl        groups containing two or more fluorine atoms, heteroaryl,        substituted phenyl, and cyano, wherein if present as sole        substituent the cyano group is located on the proximal        cyclopentadienyl ring, and may be the same as or different from        Fc;    -   X is a spacer    -   Y is a spacer    -   Z is a spacer;    -   L-F′ represents a linking moiety; and    -   [S] represents a substrate.

The linking moiety -L-F′— is in general a moiety derivable from themoiety -L-F according to general formula III or a moiety derivable fromthe moiety —R according to general formula I. In an embodiment thelinking moiety -L-F′— is a moiety derivable from the moiety -L-Faccording to general formula III.

By analogy, the invention also provides a labelled substrate for use inan electrochemical assay, the labelled substrate being of generalformula IIIb:

A-L-R′—[S]  IIIb

where R′ is a residue of R formed when reacting a compound of theinvention with a substrate.

The invention further provides assays comprising substrates according tothe invention.

DETAILED DESCRIPTION

Except where it is clear that the contrary is intended, referencesherein to “alkyl” are to straight- or branched-chain alkyl groupspreferably having from 1 to 6 carbon atoms, more preferably from 1 to 4carbon atoms, optionally interrupted by a heteroatom selected from O, Sand N and/or optionally having one or more substituents; or tocycloalkyl groups. Illustrative alkyl groups include, for example,methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl.

References herein to “cycloalkyl” are to cycloalkyl groups having up toeight, preferably up to six, ring atoms, optionally including one ormore heteroatoms. Illustrative cycloalkyl groups include, for example,cyclohexyl and heterocyclic groups such as piperidinyl and morpholinyl.

References herein to “alkenyl” are to straight- or branched-chainalkenyl groups preferably having from 1 to 6 carbon atoms, morepreferably from 1 to 4 carbon atoms, optionally having one or moresubstituents. Illustrative alkenyl groups include, for example, ethenyl,propenyl, butenyl.

The term “haloalkyl” is used herein, except where the contrary isindicated, to refer to alkyl groups having one or more halogen atomspresent as substituents, said one or more halogen atoms being selectedfrom fluorine, chlorine, bromine and iodine.

Unless the contrary is indicated, “sulfur-containing group” will beunderstood as including, without limitation, substituent groupsincluding an —S(O)₂— moiety (referred to herein as “sulfonyl”), an—S(O)— moiety (referred to herein as “sulfinyl”) or an —S— moiety(referred to herein as “sulfenyl”). Preferred sulfur-containing groupsthat may be present as substituents on the ferrocenyl rings inaccordance with the invention are those in which the sulfur atom isdirectly bonded to a ring carbon.

Unless the contrary is indicated, “phosphorus-containing group” will beunderstood as including, without limitation, substituent groupsincluding those based on phosphines or phosphine oxides, moreparticularly phosphanyl (>P—) and phosphinyl (>P(O)—) groups. Preferredphosphorus-containing groups that may be present as substituents on theferrocenyl rings in accordance with the invention are those in which thephosphorus atom is directly bonded to a ring carbon.

References herein to “heteroaryl” are to be understood as including anysingle or fused aromatic moiety including one or more heteroatoms, theheteroatoms preferably being selected from oxygen, sulphur and nitrogen.Where more than one heteroatom is present the heteroatoms may be thesame or different, each advantageously being independently selected fromoxygen, sulphur and nitrogen. Illustrative heteroaryl groups includewithout limitation furanyl, imidazolyl, thiazolyl.

References herein to “aryl” are to be understood as including any singleor fused aromatic ring system and include both hetero and other ringsystems.

The expression “substituted phenyl” as used herein includes any phenylgroup having one or more substituents attached to the phenyl ring, atany ring carbon atom of the ring. Where there is more than onesubstituent on the phenyl ring those substituents may be the same or maybe different from one another.

The application of electrochemical detection has a number of advantagesover fluorescent detection. Electrochemical detection has the potentialfor very high levels of sensitivity and exhibits a wider linear dynamicrange than fluorescence. There is no requirement for samples to beoptically clear. There is also less interference from backgroundcontaminants (many biological samples auto-fluoresce).

Electrochemical detection is based on the observation that anelectrochemically active marker exhibits different electrochemicalcharacteristics depending on whether or not it is attached to asubstrate and on the nature of the substrate. For example, in the caseof an electrochemical label attached to an amino acid, the exhibitedcharacteristics will depend not only on the identity of the amino acidbut also on whether or not that amino acid residue is incorporated intoa peptide or protein, and on the length of any such peptide or protein.Under appropriate circumstances, the electrochemical activity of amarker attached to an amino acid residue can change by a detectabledegree following loss of attachment of a single or very few amino acidresidues.

The size and characteristics of a molecule to which an electrochemicallyactive marker is attached influence the observable characteristics ofthe electrochemical marker. That may occur, for example, by influencingthe rate of migration of the marker by diffusion or its rate ofmigration in response to an electric field.

Electrochemical activity of a marker may also be influenced by stericeffects resulting from the presence of the molecule to which it islinked. For example, steric hindrance may prevent the marker fromapproaching an electrode and accepting or donating electrons.

If the marker is attached to a peptide then the secondary structure ofthe peptide (as largely determined by the primary sequence) mayinfluence the physical properties of the marker. For example, if themarker is attached to an amino acid residue in a peptide such that thestructure of the peptide sterically hinders the electrochemically activemarker then the signals observable by voltammetry may be reduced.Digestion of the peptide may destroy or release secondary structureelements and thus reduce or abolish the influence of the peptidestructure on the marker. Accordingly, digestion of the peptide resultsin a change, usually an increase, in the electrochemical signal producedby the marker moiety. In a differential pulse voltammetry experiment,the Faradaic current response at a particular applied voltage mayincrease upon digestion of the peptide.

Analogously, if a marker is attached to a nucleotide, theelectrochemical characteristics will be influenced by whether or not thenucleotide is incorporated into an oligonucleotide, upon the length ofthat oligonucleotide, and upon the sequence of the oligonucleotideespecially in the vicinity of the point of attachment.

The information relating to the electrochemically active marker can beobtained by voltammetry or by an amperometric method. Differential pulsevoltammetry is particularly suitable. If desired, the electrochemicaldetection step may be carried out using one or more electrodes coveredby a membrane which is able selectively to exclude molecules based onone or more characteristics, for example, size, charge orhydrophobicity. That may assist in eliminating background noise currentarising from, for example, charged species in the solution.

In the compounds (including labelling compounds, functionalisedlabelling compounds and labelled substrates) used in accordance with theinvention, including the compounds according to general formulae I, IIand III, the labelled substrate of general formula IIIa and the labelmoiety of general formula Ia, the two ferrocenyl groups Fc and Fc′ areeach independently selected from substituted ferrocenyl groups havingone or more substituents as defined above with reference to generalformula I. One or both pentadienyl rings of one or each of theferrocenyl moieties may be substituted by one or more substituents, thenature and location of which are selected so as to influence in adesired manner the redox characteristics of the ferrocene moiety. Inaddition to the substituents defined above, the pentadienyl rings of theferrocenyl moiety may further be substituted by any further ringsubstituent(s) that do not materially reduce the electrochemicalsensitivity of the label, or by any further ring substituent(s) thatwill enhance the electrochemical or other characteristics of the labelin any respect.

In a preferred embodiment, Fc and Fc′ are the same and each comprise atleast one substituent selected from the group consisting of:

-   -   sulfur-containing groups selected from sulfenyl, sulfinyl and        sulfonyl groups, phosphorus-containing groups selected from        phosphanyl and phosphinyl groups,    -   iodo,    -   chloro,    -   fluoroalkyl groups containing two or more fluorine atoms,        especially trifluoroalkyl,    -   heteroaryl, and    -   substituted phenyl.

In another embodiment, Fc and Fc′ are the same and there is assubstituent a cyano group located on the respective proximalcyclopentadienyl ring of each of said Fc and Fc′ moieties.

In one preferred embodiment of the invention, Fc and Fc′ are the sameand each comprise at least one ring substituent selected fromsulfur-containing groups and phosphorus-containing groups. In oneillustrative Example below from the group consisting ofsulfur-containing groups and phosphorus-containing groups, there isdisclosed a compound with an electrochemical potential value of inexcess of 500 mV. It is believed that compounds of the invention thatinclude sulfur-containing groups or phosphorus-containing groups,especially those in which the sulfur or phosphorus atom is directlyboded to a ring carbon of the ferrocenyl, will be advantageous inextending the range of available potential values of such labels, makingthem useful as labels in electrochemical assays. In particular, thosecompounds offer the possibility of use in assays in which the highelectrochemical potential value may be valuable, for example inmultiplex assays where a range of different labels with differentiableelectrochemical potentials are used.

In certain preferred compounds there is present as a saidsulfur-containing group at least one sulfonyl substituent selected fromgroups of the formula R¹⁵S(O)₂—, wherein R¹⁵ is selected from branched-or straight-chain alkyl, haloalkyl, and substituted or unsubstitutedaryl. Illustrative alkyl groups R¹⁵ include, for example, methyl, ethyl,propyl, or butyl, especially t-butyl. Preferred haloalkyl groups R¹⁵include, for example, fluoroalkyl groups with one of more fluorosubstituents, especially trifluoromethyl. In certain preferredembodiments, R¹⁵ represents unsubstituted C1 to C4 alkyl; or C1 to C4-haloalkyl, for example C1 to C4- fluoroalkyl, especiallytrifluoromethyl. Illustrative aryl groups R¹⁵ include, especially,phenyl, which may be substituted or unsubstituted, with preferredsubstituents including, for example, halo, unsubstituted alkyl(preferably C1 to C4 alkyl), substituted alkyl (for example haloalkyl),nitro, cyano, alkoxy (for example, C1 to C4 alkoxy, preferably methoxy)and sulfur-containing groups, for example sulfonyl. Other illustrativearyl substituents R¹⁵ include heteroaryl groups containing at least oneheteroatom selected from oxygen, sulphur and nitrogen. Illustrative ofpreferred aryl groups R¹⁵ are those of general formula (R¹⁶)_(a)—Ar—, or(R¹⁶)_(a)— HeAr— in which Ar represents aryl; HeAr representsheteroaryl; R¹⁶ is a substituent selected from halo, alkyl, nitro,cyano, haloalkyl, alkoxy, and sulphur-containing groups, for examplesulfonyl; and a is an integer in the range of from 0 to a number equalto the maximum substitutable ring positions in the aryl, or heteroarylring. For example, R¹⁵ may represent phenyl substituted by F; Cl; Br; I;unsubstituted C1 to C4 alkyl; C1 to C4 haloalkyl, for exampletrifluoromethyl; nitro; cyano; methoxy; or sulfur-containing groups, forexample sulfonyl.

In further embodiments, there is present as a said phosphorus-containingsubstituent a group of the general formula (R¹⁷)₂P(O)—, wherein each R¹⁷is independently selected from branched- or straight-chain alkyl,haloalkyl, substituted or unsubstituted aryl and substituted orunsubstituted heteroaryl. Advantageously, R¹⁷ represents C1 to C4 alkyl,which is preferably unsubstituted, for example, methyl, ethyl, propyl,or butyl, especially t-butyl.

Illustrative aryl substituents R¹⁷ include phenyl and heteroaryl groupscontaining at least one heteroatom selected from oxygen, sulphur andnitrogen, each of which may be unsubstituted or substituted. Preferredaryl groups R¹⁷ include those of general formula (R¹⁸)_(b)— Ar—, or(R¹⁸)_(b)—HeAr— in which Ar is aryl; HeAr is heteroaryl; R¹⁸ is asubstituent selected from halo, alkyl, nitro, cyano, haloalkyl, alkoxyand sulphur-containing groups, for example sulfonyl; and b is an integerin the range of from 0 to a number equal to the maximum substitutablering positions in the aryl or heteroaryl ring. For example, R¹⁷ mayrepresent phenyl substituted by F, Cl, Br, I, unsubstituted C1 to C4alkyl, C1 to C4 haloalkyl, for example trifluoromethyl, nitro, cyano, ormethoxy. Preferably, Ar represents phenyl and comprises one or moresubstituents R¹⁸ (which may be the same or different) selected fromhalo, alkyl, haloalkyl, nitro, cyano, and alkoxy. More preferably, eachR¹⁷ is the same and represents phenyl with at least one substituent,especially phenyl with one substituent in the 4-position. In onepreferred embodiment, R¹⁷ represents branched C1 to C4 alkyl, forexample t-butyl.

In another advantageous embodiment the ferrocenyl groups are the sameand there is present as a said substituent on each ferrocenyl at leastone substituted phenyl group in which the phenyl has at least onesubstituent selected from halo, C1 to C4 alkyl, nitro, cyano, C1 to C4haloalkyl, C1 to C4 alkoxy, and sulfur-containing radicals, for example,sulfonyl. Illustrative of such substituents on the phenyl are, forexample, fluorine, chlorine, bromine, iodine atoms, nitro, cyano,trifluoromethyl, and methoxy. In certain embodiments, each phenyl hasone substituent which may be located in the 4-position, for example,4-nitrophenyl (wherein the ferrocenyl group is attached to the phenylgroup at the 1-position).

In a further embodiment there is present as a said substituent on eachferrocenyl at least one heteroaryl group which may be unsubstituted orsubstituted by at least one substituent selected from halo, C1 to C4alkyl, nitro, cyano, C1 to C4 haloalkyl and C1 to C4 alkoxy. Forexample, there may be present as a heteroaryl group furanyl.

In yet further embodiments, there may be present as a said substituenton each ferrocenyl at least one iodine or chlorine atom. Other possiblesubstituents include at least one silyl substituent, preferably a silylgroup selected from alkyl silyl groups, for example trialkylsilyl,especially trimethylsilyl.

In addition to at least one substituent as defined with reference togeneral formula I herein, each ferrocenyl moiety may optionally befurther substituted by at least one additional substituent, for example,by at least one additional substituent selected from bromo, fluoro, C1to C4-alkyl, haloalkyl, and C1 to C4 alkenyl. In accordance with afurther embodiment, Fc and Fc′ may each additionally comprise at leastone cyano group substituent on its distal ring.

It is preferred that the ferrocenyl moieties are identical. That isthought to give a stronger signal.

The moiety Z may be unsubstituted or substituted. Substituents, whenpresent, may be for example one or more substituents selected fromhydroxy, halo, cyano, amino, and unsubstituted or substituted C1-C4alkyl, C1-C4 alkenyl, or aryl; wherein in each case optionalsubstituents include without limitation hydroxy, halo, cyano, oxo,amino, ester or amido. The moiety Z may, if desired, be interrupted byone, or optionally more than one, atom or moiety selected from —O—, —S—,cycloalkyl, including heterocycloalkyl, —CO—, —CONH—, —NHCO— and —NH—and —NR¹— in which R¹ is C1 to C4 alkyl. Illustrative of cycloalkylmoieties that may be included as interruptions within the moiety Z arecycloalkyl rings with from 5 to 7 ring atoms, especially 6 ring atoms,for example cyclohexyl, piperidinyl, morpholinyl.

The moieties X and Y, which are preferably the same, advantageously havea chain length of from 1 to 6, preferably from 1 to 4 carbon atoms,especially one or two carbon atoms, and more especially one carbon atom.The moieties X and Y may each represent an alkylene chain, optionallyinterrupted by —O—, —S— or —NR⁵—for example —NH—. Preferred moieties Xand Y include, for example, —CH₂—, —CH₂— CH₂—, —(CH₂)₃— O—CH₂—, —CH₂—O—(CH₂)₃—, —(CH₂)₃— O—(CH₂)₂—, and —(CH₂)₂— O—(CH₂)₃—.

In some embodiments, labels according to the present invention may beprepared by reacting two equivalents of a suitable substituted ferrocenecarboxaldehyde in a suitable solvent in the presence of a reducingagent. The structure of the desired label, including the structure ofmoiety Z, may be determined by selection of suitable starting materialsand/or routine modification of the synthesis method. In one illustrativemethod, for example a ferrocene derivative such as 1′-iodo ferrocenecarboxaldehyde, 1′-chloro ferrocene carboxaldehyde, 1′-furanyl ferrocenecarboxaldehyde or 2-tert-butyl sulphonyl ferrocene carboxaldehyde may bereacted with a suitable amine (for example, 6-aminohexan-1-ol, glycineor (aminoethoxy)ethanol)) in a suitable solvent, for example THF, in thepresence of a reducing agent, for example sodium triacetoxyborohydride.When glycine is used as the amine, the resulting di-ferrocenyl glycinederivative may be further modified to generate a desired structure. Forexample, it may be reacted with oxalyl chloride in dichloromethane thentreated with 4-(hydroxymethyl)piperidine to generate aferrocene-substituted derivative of2-((di-ferrocenylmethyl)amino)-1-(4-(hydroxymethyl)piperidin-1-yl)ethanone.In another embodiment, when glycine is used as the amine, the resultingdi-ferrocenyl glycine derivative may be further reacted with oxalylchloride in dichloromethane then treated with 6-aminohexan-1-ol togenerate a ferrocene-substituted derivative ofN,N-2-(diferrocenylmethylamino)acetyl-6-aminohexanol (also namedN-(6-hydroxylhexyl)-2-((diferrocenylmethyl)amino)-acetamide). Suitablemethods for synthesis of other compounds according to the invention willbe apparent to those skilled in the art in the light of the disclosureherein.

Linkage to the substrate can be by any suitable linkage, typically bylinkage to a substrate side chain. The linker group R in the compoundsof general formula I may be any group suitable for effecting linkage tothe substrate either directly or via a functionalising group asdescribed herein. R is advantageously, although not necessarily, alinker group comprising an oxygen atom. R is preferably a hydroxyl groupor a protected hydroxyl group or a group containing a hydroxyl group ora protected hydroxyl group. It will be appreciated, however, that anyother suitable linker group R may be selected having regard to thesubstrate to which, in use, the compound is to be attached. Varioussynthetic methods have been developed for the derivatisation of protein,peptide or amino acid side chains or protein, peptide or amino acidterminal moieties. For example, lysine residues in a protein may bederivatised by reaction with a succinimidyl ester. For derivatisation atother amino acid residues, other known synthetic methods may be used.For example, a maleimide reagent may be used to derivatise cysteineresidues. An N-hydroxy succinimide ester may be used to derivatise theamino terminus or side chain amino group of a protein or peptide, or anamino moiety of an amino acid.

Suitable derivatisation methods for nucleotides are also well-known, forexample, using a phosphoramidite moiety.

The above derivatisation methods are illustrative of the methods thatmay be used to link the compounds of the invention to a substrate,although other methods may be used.

Labelled substrates according to the invention may be prepared byreaction of a compound according to the invention, optionally afterfunctionalisation to obtain a functionalised labelling compound, with asubstrate, for example, with a substrate selected from amino acids,nucleotides (for example oligo deoxyribonucleotides or oligoribonucleotides), nucleosides, sugars, peptides, proteins,oligonucleotides, polynucleotides, carbohydrates and derivatives of anyof those molecules.

In a preferred embodiment, the substrate is a nucleotide or anoligonucleotide. The nucleotide may be selected from adenosine,thymidine, guanosine, cytidine or uridine nucleotides. Preferably thenucleotide, or a nucleotide of the oligonucleotide, is attached to thelabel through a group attached to the ribose or deoxyribose group of thenucleotide, for example in the 2′, 3′ or 5′ position, for examplethrough an oxygen or nitrogen atom. Most preferably, the nucleotide isattached at the 3′ or 5′ position, for example at the 5′ position.Linking at other positions is also possible.

In the case of nucleotides, one advantageous way of attaching labels ofthe invention is by functionalization with phosphoramidite. The linkingof phosphoramidite groups to oligonucleotides is widely practised inoligonucleotide synthesis and thus methods and conditions for attachmentto an oligonucleotide of labels functionalised with phosphoramidite willbe well-known and a routine matter to those skilled in the art. Further,it advantageously permits the use of standard oligo manufacturingmethods.

Oligonucleotides for use in an assay in accordance with the inventionare advantageously nucleotides having from 2 to 50 nucleotides, morepreferably from 2 to 40 nucleotides especially from 15 to 35nucleotides, with from 18 to 30 nucleotides being especially preferred.For some applications, shorter oligonucleotides may be useful, forexample oligonucleotides with from 2 to 14 nucleotides, more preferablyfrom 2 to 10 nucleotides.

Attachment to proteins, for example via cysteine or lysine, may beaccomplished in some cases by incubation of the protein and ferrocenyllabel together at room temperature in an appropriate buffer solution.Where the label is advantageously to be linked to cysteine or lysine butthe substrate sequence does not contain cysteine or lysine at a suitableposition the sequence may if desired be mutated to add one or morecysteine or lysine residue either as an additional residue or as asubstitution for another residue. An alternative method for attachmentto proteins may include biotinylation of the labels and use ofcommercial streptavidinated proteins (or vice versa). By way of example,the substrate may be biotinylated by any standard technique for exampleby use of a commercially available biotinylation kit. Biotinylatedsubstrate will bind to strepavidin or avidin conjugated compounds suchas antibodies (which are commercially and widely available).

It will however be apparent to the skilled person that similar labelsmay be attached to a substrate at a selected one of a number oflocations by use of an appropriate labelling functional group.

In functionalised labelling compounds of the general formula III:

A-L-F  III

A-L is preferably a moiety derived from a compound according to generalformula I and F is a functionalising group. Preferred functionalisedlabelling compounds of the general formula III include compounds of thegeneral formula IIIb:

A-O—F  IIIb

wherein A-O is a moiety derived from a compound according to generalformula I, preferably by loss of a hydroxy hydrogen atom or protectinggroup when the linker group R of general formula I is hydroxyl or ahydroxyl-containing group or is a protected hydroxyl group, and F is afunctionalising group.

Suitable functionalising groups that may be usable with labels of theinvention, including as functionalising group F in general formula IIIand general formula IIIb, may include, without limitation, succinimidylester groups, phosphoramidite groups, maleimide groups, biotin and azidegroups. It will be appreciated, however, that there may be used anyfunctionalising group that facilitates attachment of the labellingcompound to the substrate to be labelled.

The invention also provides a method of detecting a nucleic acid (forexample RNA or DNA) in a sample comprising the optional step ofamplifying the nucleic acid (for example by PCR or another nucleic acidamplification technique) followed by the step of contacting the amplicon(or the nucleic acid) with a complementary nucleic acid probe underconditions to allow hybridization between the probe and amplicon (or thenucleic acid), followed by the step of selectively degrading eitherhybridized or unhybridized probe (for example by use of single or doublestrand specific nucleases), wherein said probe is labelled with anelectrochemically active compound of the invention and wherein themethod provides the step of measuring the electrochemical activity ofthe compound labelling the probe of wherein said electrochemicalactivity is dependent either quantitatively or qualitatively on theextent of degradation of the probe.

The invention also provides a method of detecting an antibody orderivative (which may for example be bound to target antigen in anassay) with an electrochemically active compound of the inventioncomprising the step of measuring the electrochemical activity of thecompound. This method can be performed quantitatively or qualitatively.

The invention also provides methods of diagnosing or monitoring adisease in a subject comprising using a method of the invention in thedetection of a protease or a protease inhibitor associated with saiddisease in a tissue or body fluid of the subject. A substrate for theprotease can be labelled according to the invention.

The invention also provides methods of diagnosing or maintaining adisease in a subject comprising using a method of the invention todetect a peptide or protein associated with said disease in a tissue orbody fluid of the subject.

The invention also provides methods of diagnosing or monitoring adisease in a subject comprising using a method of the invention in thedetection of a nuclease or a nuclease inhibitor associated with saiddisease in a tissue or body fluid of the subject.

Furthermore, the invention provides use of a method of the invention fordetecting a disease in a subject.

The invention also provides methods of detecting a microorganism (inparticular, a pathogen or other undesirable organism, for example a foodspoilage organism), comprising using a method of the invention. Asubstrate from the microorganism (or derived from the pathogen e.g. anucleic acid amplicon produced using a target nucleic acid sequence inthe pathogen) can be labelled according to the invention. Detection ofthe labelled substrate can be used to indicate detection of themicroorganism.

The invention also provides an assay comprising a step which uses alabelled substrate of the invention, optionally in combination withother assay components for example a sample vessel, a containercomprising electrodes for electrochemical detection, enzymes for use inthe assay or standards and controls. Said assay may use more than onedifferent labelled substrate of the invention. If that is the case thepresence of different labelled substrates may be differentially detectedby labelling them with electrochemical labels of the invention havingdifferent electrochemical characteristics (for example differentoxidation potentials) thereby permitting the assay to be a multiplex(for example a duplex) assay in which different substrates may bediscriminated when present in the same sample vessel. Simplex assays arealso encompassed by the invention.

Table 1a below sets out certain illustrative formulae of compoundsaccording to the invention which may be used as labels inelectrochemical assays in accordance with the invention, and which maybe used to make functionalised labelling compounds and labelledsubstrates according to the invention. Table 1a also sets out in thesecond column illustrative corresponding functionalised labellingcompounds according to the invention. Tables 1b, 2, 3 and 4 set outgeneral formulae of further illustrative compounds of the invention.Whilst functionalised compounds corresponding to the compoundsidentified in Tables 1b, 2, 3 and 4 are not shown, it will beappreciated that the compounds shown may be functionalised by additionof any suitable functionalising moiety. In the formulae in Tables 1a,1b, and 2 to 4, except where considerations of steric hindrance mitigateagainst it, each ferrocenyl may have more than one substituent, whichmay be the same or different, and in any ring position. Both ferrocenylgroups preferably have the same substituent(s) in the same positions,i.e. both ferrocenyl groups are the same.

TABLE 1a Illustrative embodiments with cyclopentadienyl ringsubstituents in accordance with the invention General formula ofcompound General formula-illustrative functionalised labelIdentification of symbols 1

R¹⁰ represents a radical selected from S-containing groups, P-containinggroups, I, Cl, trialkylsilyl, CF₃, heteroaryl, substituted phenyl; qrepresents from 1 to 5, for example 1; and W represents (CH₂)_(n) wheren is from 0 to 6, O, S or NR²⁰ where R²⁰ is alkyl, for example Cl to C4alkyl 2

R¹¹ represents a radical selected from S-containing groups, P-containinggroups, I, Cl, trialkylsilyl, CF₃, heteroaryl, substituted phenyl andcyano; r represents from 1 to 5, for example 1; and W represents(CH₂)_(n) where n is from 0 to 6, 0, S or NR²⁰ where R²⁰ is alkyl, forexample C1 to C4 alkyl 3

R¹² represents a radical selected from S-containing groups, P-containinggroups, I, Cl, trialkylsilyl, CF₃, heteroaryl, substituted phenyl; qrepresents from 1 to 5, for example 1; and W represents (CH₂)_(n) wheren is from 0 to 6, O, S or NR²⁰ where R²⁰ is alkyl, for example C1 to C4alkyl 4

R¹³ represents a radical selected from S-containing groups, P-containinggroups, I, Cl, trialkylsilyl, CF₃, heteroaryl, substituted phenyl; qrepresents from 1 to 5, for example 1; and W represents (CH₂)_(n) wheren is from 0 to 6, O, S or NR²⁰ where R²⁰ is alkyl, for example C1 to C4alkyl 5

R¹⁴ represents a radical selected from S-containing groups, P-containinggroups, I, Cl, trialkylsilyl, CF₃, heteroaryl, substituted phenyl; qrepresents from 1 to 5, for example 1; W represents (CH₂)_(n) where n isfrom 0 to 6, O, S or NR²⁰ where R²⁰ is alkyl, for example C1 to C4alkyl; and V represents (CH₂)_(m) where m represents from 2 to 6,optionally interrupted by an O atom

TABLE 1b Further illustrative embodiments with cyclopentadienyl ringsubstituents in accordance with the invention General formula ofcompound Identification of symbols 6

R¹² represents a radical selected from S- containing groups,P-containing groups, I, Cl, trialkylsilyl, CF₃, heteroaryl, substitutedphenyl, cyano; q represents from 1 to 4, for example 1; and W represents(CH₂)_(n) where n is from 0 to 6, O, S or NR²⁰ where R²⁰ is alkyl, forexample C1 to C4 alkyl 7

R¹³ represents a radical selected from S- containing groups,P-containing groups, I, Cl, trialkylsilyl, CF₃, heteroaryl, substitutedphenyl, cyano; q represents from 1 to 4, for example 1; and W represents(CH₂)_(n) where n is from 0 to 6, O, S or NR²⁰ where R²⁰ is alkyl, forexample C1 to C4 alkyl 8

R¹⁴ represents a radical selected from S- containing groups,P-containing groups, I, Cl, trialkylsilyl, CF₃, heteroaryl, substitutedphenyl, cyano; q represents from 1 to 5, for example 1; W represents(CH₂)_(n) where n is from 0 to 6, O, S or NR²⁰ where R²⁰ is alkyl, forexample C1 to C4 alkyl; and V represents (CH₂)_(m) where m representsfrom 2 to 6 optionally interrupted by an O atom

The compounds 6 to 8 above may be functionalised by any suitable method,for example by phosphoramidation analogously to the compounds 1 to 5shown in Table 1 above.

In Table 2 below there are shown general formulae describing certainpreferred embodiments of the invention in which each ferrocenyl group issubstituted by a sulfonyl group. Table 3 below shows general formulaedescribing certain preferred embodiments of the invention in which eachferrocenyl group is substituted by a phosphinyl group. The compounds inTables 2 and 3 may each be functionalised by any suitable method, forexample phosphoramidation. The present invention encompasses thefunctionalised analogs of the compounds defined in the Tables 1a, 1b, 2and 3 as well as labelled substrates derived therefrom.

TABLE 2 Illustrative embodiments with sulfonyl radicals ascyclopentadienyl ring substituents

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Where present in the formulae 9 to 20 above:

-   -   R¹⁵ represents alkyl, for example t-butyl, haloalkyl, especially        fluoroalkyl, for example CF₃, phenyl or substituted phenyl;    -   W represents (CH₂)_(n) where n is from 0 to 6, O, S or NR²⁰        where R²⁰ is alkyl, for example C1 to C4 alkyl; and    -   V, where present, represents (CH₂)_(m) where m represents from 2        to 6, optionally interrupted by an O atom; and    -   R¹⁶, where present, represents one or more radicals selected        from F, Cl, Br, I, alkyl, NO₂, cyano, CF₃ and methoxy.

TABLE 3 Illustrative embodiments with phosphinyl radicals ascyclopentadienyl ring substituents

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

Where present in the respective formulae 21 to 32 above:

-   -   R¹⁷ represents alkyl, for example t-butyl, phenyl or substituted        phenyl;    -   W represents (CH₂)_(n) where n is from 0 to 6, O, S or NR²⁰        where R²⁰ is alkyl, for example C1 to C4 alkyl;    -   V represents (CH₂)_(m) where m represents from 2 to 6,        optionally interrupted by an O atom; and    -   R¹⁸ represents one or more radicals selected from F, Cl, Br, I,        alkyl, NO₂, cyano, CF₃ and methoxy; in an especially preferred        embodiment, R¹⁸ represents one of said radicals and is located        in the 4-position.

In the general formulae and their functionalised counterparts in Tables1a, 1b, 2 and 3 when one or more ring substituents is present on theproximal pentadienyl ring of each ferrocenyl, that is, the ring that isdirectly bonded to the rest of the molecule, there is preferably a saidring substituent at an adjacent ring position to that bond. When morethan one ring substituent is present on each proximal pentadienyl ring,those substituents may be in any position relative to one another. Whenmore than one ring substituent is present on each distal pentadienylring of each ferrocenyl, that is, the ring remote from the bond linkingthe ferrocenyl to the rest of the molecule, those substituents may be inany position relative to one another. Whilst in the compounds shown inthe above Tables there are shown ring substituents on either theproximal or the distal ring, it is also possible for both pentadienylrings of each ferrocenyl to carry one or more substituents.

As illustrated in the Examples herein, incorporation of one or moresubstituents on each of the ferrocenyl groups (the substituents on eachferrocenyl being the same) can be used to obtain compounds with modifiedelectrochemical characteristics, providing through appropriatesubstituent selection a suite of compounds from which two or more may beselected for the purpose of multiplex reactions.

Certain illustrative compounds according to the invention which havebeen found to have good electrochemical properties are set out in Table4 below. The invention includes in addition to the compounds in Table 4functionalised labelling compounds and labelled substrates which arederivable from those compounds in accordance with the invention.

TABLE 4 Illustrative labelling compounds according to the invention

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The following examples illustrate compounds of the invention wherein Fcand Fc′ are the same:

Materials and Methods

-   1′-Iodo ferrocene carboxaldehyde was synthesised from iodoferrocene    using the method described in Organometallics, 2011, 30, 3504-3511.-   1′-Furanyl ferrocene carboxaldehyde was synthesised from    1′-iodoferrocene carboxaldehyde, using method adapted from    Angewandte Chemie, 2006, 45, 1282-1284.-   1-[(Dimethylamino)methyl]-2-(di-tert-butyl phosphinyl)-ferrocene was    synthesised from dimethylaminomethyl ferrocene using method adapted    from Organometallics, 1985, 7 1297-1302.-   1′-Chloro ferrocene carboxaldehyde was prepared from chloroferrocene    using the procedure from Coll. Chechoslovak. Chemm. Commun., 1987,    52, 174-181.-   2-tert-Butyl sulphonyl ferrocene carboxaldehyde was obtained from    synthesised from dimethylaminomethyl ferrocene using method adapted    from Organometallics, 1985, 7, 1297-1302.-   2-Cyanoethyldiisopropylchlorophosphoramidite was obtained from    Sigma-Aldrich.-   6-(Bis((2-formyl)1-methylferrocenyl)amino)hexan-1-ol was synthesised    from (+/−)-4-(methoxymethyl)-2-ferrocenyl-1,3-dioxane using a method    adapted from Journal of Organic Chemistry, 1997, 62, 6733-6745.-   Iodoferrocene was synthesised from ferrocene, from an adaptation of    the method described in Journal of Organometallic Chemistry, 2011,    696, 1536-1540, utilising iodine as a suitable electrophile.-   Chloroferrocene prepared from ferrocene using a modified procedure    from J. Organomet. Chem., 1996, 512, 219-224, using hexachloroethane    as a chlorinating reagent.-   6-Aminohexanol, ferrocene, dimethylaminomethyl ferrocene and    4-nitrobenzene boronic acid were obtained from Sigma-Aldrich.-   1-[(Dimethylamino)methyl]-2-(t-butylthio)-ferrocene was prepared    using the procedure from Organomet., 1988, 7, 1297-1302.-   3-tert-butylsulfinyl ferrocene carboxaldehyde was prepared according    to the procedure described in Chem. Commun., 2004, 598-599.

Determination of Electrochemical Potential

The electrochemical potential values mentioned hereafter were measuredusing an electrochemical cell including as background electrolyte anaqueous 100 mM solution of sodium chloride, using a printed carbonworking electrode, a printed carbon counter electrode and asilver/silver chloride reference electrode, all with silver connectors.The electrodes were ink based and were screen printed on to a polymersubstrate (for example Mylar) followed by heat curing. By way ofillustration, the sample may be prepared as follows: Ferrocenyl labelprecursor (2 ng) is dissolved in DMSO (1 mL). An aliquot of 10 μL istaken of this solution and is then further diluted in the buffer (500μL). Then an aliquot (20 μL) is applied to the screen printed electrodeto run the electrochemical scan. An illustrative form of suitable cellis described and shown schematically in WO2012/085591.

Example 1—Synthesis of 6-(bis((1′-iodoferrocenyl)1-methylferrocenyl)amino)hexan-1-ol

1′-Iodo ferrocene carboxaldehyde (259 mg, 0.76 mmol) was dissolved indry THF (7 cm³) and treated with 6-aminohexan-1-ol (44 mg, 0.38 mmol)and sodium trisacetoxyborohydride (313 mg, 1.91 mmol) successively. Thesolution was allowed to stir at room temperature overnight. After thistime the reaction was quenched by addition of NaHCO₃ (sat) (10 cm³). Theorganic layer was separated, then the aqueous layer back extracted withEtOAc (3×10 cm³). Combined organic extracts were dried over MgSO₄,filtered then concentrated in vacuo to give an orange oil. Product waspurified by silica chromatography, eluting with 1:1 (EtOAc:Hexane)+1%NH₃OH. To give the desired product as an orange oil 123 mg, 42%. ¹H NMR(300 MHz; d₆-benzene) δ_(H): 4.28 (4H, t, J 1.8, F_(c)H), 4.14 (4H, t, J1.5, F_(c)H), 4.11 (4H, t, J 1.5, F_(c)H), 4.06 (4H, t, J 1.8, F_(c)H),3.60 (2H, t, J 6.6, CH₂O), 3.41 (4H, s, F_(c)CH₂N), 2.27 (2H, t, J7.4,NCH₂), 1.55-1.23 (8H, m, CH₂); ¹³C NMR (75 Mhz; d₆-benzene) δ_(C): 85.2,75.1, 73.3, 70.9, 69.4, 62.98, 52.02, 51.8, 40.5, 32.7, 27.1, 27.0,25.5; HRMS (ESI) m/z calcd for C₂₈H₃₃NOFe₂I₂ 765.9428, m/z found765.9460.

The electrochemical potential was measured and found to be 442 mV.

Example 2—Synthesis of6-(bis((1′-(4-nitrophenyl)ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol

To a Schlenk tube was added 4-nitrobenzene boronic acid (39.6 mg, 0.23mmol), trisdibenzylideneacetone palladium (Pd₂(dba)₃- 1.9 mg, 2 mol %),tricyclohexylphosphine (1.4 mg, 4.8 mol %). The flask was sealed andevacuated and back filled with argon four times. The6-(bis((1′-iodoferrocenyl) 1-methylferrocenyl)amino)hexan-1-ol (83 mg,0.11 mmol) in 1,4-dioxane (2 cm³) was then added to the flask. This wasthen followed by 1.27 M K₃PO₄ (aq) (141 μl, 0.18 mmol). The flask wasthen heated at 100° C. overnight. After this time the reaction wasallowed to cool to room temperature then diluted with EtOAc (5 cm³) andH₂O (5 cm³). The organic layer was separated and the aqueous layer backextracted with EtOAc (3×5 cm³). The combined organics were then washedwith brine (sat) (10 cm³), dried over Na₂SO₄, filtered and concentratedin vacuo to give a purple solid. Product was purified by silicachromatography, eluting with EtOAc+1% NH₃OH to give the desired productas an amorphous solid 11 mg, 13%. ¹H NMR (300 Mhz; d₆-benzene) δ_(H):8.05 (4H, app d, J 8.9, ArH), 7.08 (4H, app t, J 8.9, ArH), 4.39 (4H, t,J 1.8, F_(c)H), 4.24 (4H, t, J 1.8, F_(c)H), 3.96 (4H, t, J 1.8,F_(c)H), 3.88 (4H, t, J 1.8, F_(c)H), 3.52 (2H, t, J 5.8, CH₂OH), 3.01(4H, s, CH₂F_(c)), 2.28 (2H, t, J 6.9, NCH₂), 1.50-1.26 (8H, m, CH₂).

The electrochemical potential was measured and found to be 437 mV.

Example 3—Synthesis of6-(bis((1′-(1-furanyl)ferrocenyl)1-methylferrocenyl) amino)hexan-1-ol

1′-Furanyl ferrocene carboxaldehyde (34 mg, 0.12 mmol) was dissolved indry THF (1 cm³) and treated with 6-aminohexan-1-ol (7 mg, 0.06 mmol) andsodium trisacetoxyborohydride (49 mg, 0.3 mmol) successively. Thesolution was allowed to stir at room temperature overnight. After thistime the reaction was quenched by addition of NaHCO₃(sat) (5 cm³). Theorganic layer was separated, then the aqueous layer back extracted withEtOAc (3×5 cm³). Combined organic extracts were dried over MgSO₄,filtered then concentrated in vacuo to give an orange oil. Product waspurified by silica chromatography, eluting with 1:1 (EtOAc:Hexane)+1%NH₃OH to give the desired product as an orange oil 15 mg, 39%. ¹H NMR(300 Mhz; d₆-benzene) δ_(H): 7.26 (2H m, ArH), 6.25 (2H, dd, J 6.5, 1.8,ArH), 6.20 (2H, dd, J 6.5, 0.7, ArH), 4.61 (4H, t, J 1.9, F_(c)H), 4.21(4H, t, J 1.9, F_(c)H), 4.12 (4H, t, J 1.9, F_(c)H), 4.05 (4H, t, J 1.9,F_(c)H), 3.44 (2H, t, J 6.3, CH₂OH), 3.35 (4H, s, CH₂F_(c)), 2.40 (2H,t, J7.2, CH₂N), 1.56-1.27 (8H, m, CH₂), ¹³C NMR (75 Mhz; d₆-benzene)δ_(C): 154.2, 141.6, 111.9, 104.4, 86.1, 77.8, 72.2, 69.6, 66.5, 63.03,52.4, 33.5, 27.9, 27.63, 26.3, HRMS (ESI) m/z calcd forC₃₆H₃₉Fe₂NO₃668.15264 m/z found 668.1558.

The electrochemical potential was measured and determined to be 339 mV.

Example 4: Synthesis of6-(bis((2-di-tert-butyl-phosphinyl-ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol

6-(bis((2-di-tert-butyl-phosphinyl-ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol was synthesised as shown in thescheme below.

To a solution of 1-[(dimethylamino)methyl]-2-(di-tert-butylphosphinyl)-ferrocene (145 mg, 0.3 mmol) in diethyl ether (5 cm³) wasadded methyl iodide (111 μl, 1.7 mmol). The orange solution was allowedto stir at room temperature under N₂ for 1 hour. The orange suspensionthat was formed was then concentrated in vacuo to give a bright orangesolid. The bright orange solid was then taken up in dry acetonitrile (3cm³) and then treated with 6-amino-hexan-1-ol (17.5 mg, 0.15 mmol). Theflask was then sealed and heated at reflux for 18 hours. After this timethe dark orange solution was allowed to cool to room temperature. Thereaction was partitioned between CH₂Cl₂ (5 cm³) and NaHCO₃(sat) (5 cm³),the organic layer was separated and then dried over Na₂SO₄, filtered andthen concentrated under reduced pressure to give a brown oil.Purification by basic alumina chromatography eluting with 2% MeOH:CH₂Cl₂gave the desired product as an orange oil (8 mg, 7% yield)

¹H NMR (250 MHz; d₆-benzene) δ_(H) 4.63 (2H, app s, F_(c)H), 4.14 (12H,s, F_(c)H), 3.85 (6H, br s, F_(c)H+F_(c)NCH₂), 3.55 (2H, app s, OCH₂),2.45 (2H, app s, CH₂N), 1.47-0.95 (44H, m, tBu+CH₂), ³¹P NMR (121 MHz;d₆-benzene) δ_(P) 61.3, HRMS (ESI μTOF) m/z calcd for C₄₄H₆₉NO₃Fe₂P₂834.3529 m/z found 834.3603.

The electrochemical potential (DPV) was measured and found to be 512 mV.

Example 5:6-(bis((1′-chloroferrocenyl)1-methylferrocenyl)amino)hexan-1-ol

6-(bis((1′-chloroferrocenyl) 1-methylferrocenyl)amino)hexan-1-ol wassynthesised as shown in the scheme below.

1′-chloro ferrocene carboxaldehyde (1.35 g, 5.44 mmol) was dissolved indry THF (50 cm³) and treated with 6-aminohexan-1-ol (318 mg, 2.72 mmol)and sodiumtrisacetoxyborohydride (1.1 g, 6.80 mmol) successively. Thesolution was allowed to stir at room temperature overnight. After thistime the reaction was quenched by addition of NaHCO₃(sat) (30 cm³). Theorganic layer was separated, then the aqueous layer back extracted withEtOAc (3×30 cm³). Combined organic extracts were dried over MgSO₄,filtered then concentrated in vacuo to give an orange oil. Product waspurified by silica chromatography, eluting with 1:1 (EtOAc:Hexane)+1%NH₃OH to give the desired product as an orange oil 892 mg, 56%.

¹H NMR (300 MHz; d₆-benzene) δ_(H) 4.18 (4H, t, J 1.6, F_(c)H), 4.14(4H, t, J 1.8, F_(c)H), 3.98 (4H, t, J 1.6, F_(c)H), 3.65 (4H, t, J 1.8,F_(c)H), 3.65 (4H, s, NCH₂, F_(c)CH₂N), 3.34 (2H, t, J 6.3, OCH₂), 2.41(2H, t, J7.0, NCH₂), 1.55-1.23 (8H, m, CH₂), ¹³C NMR (75 MHz;d₆-benzene) δ_(C) 93.4, 86.6, 72.9, 70.6, 69.1, 68.1, 67.3, 63.0, 52.8,33.6, 28.1, 27.7, 26.3, HRMS (ESI μTOF) m/z calcd for C₂₈H₃₃NOFe₂Cl₂582.0716 m/z found 582.0722.

The electrochemical potential (DPV) was measured and found to be 452 mV.

Example 6: 6-(bis((2-cyano)1-methylferrocenyl)amino)hexan-1-ol

6-(bis((2-formyl) 1-methylferrocenyl)amino)hexan-1-ol (1 eq) isdissolved in ethanol and treated with hydroxylamine hydrochloride (5 eq)and sodium acetate (5 eq). The resulting suspension is then heated atreflux for 18 hrs. After this time the reaction is allowed to cool toroom temperature and concentrated in vacuo. The solid residue is thenpartitioned between chloroform and NaHCO₃(sat). The organic layer isseparated and dried over Na₂SO₄, filtered and concentrated in vacuo togive the corresponding oxime. The oxime is then taken up in dry THF andtreated with (benzotriazol-1-yloxy)tris(dimethylamino)phosphoniumhexafluorophosphate (BOP-2 eq) and stirred for 5 mins. Then1,8-diazabicyclo[5.4.0]undec-7-ene (2.3 eq) is added. The solution isthen stirred for 90 mins. The reaction is then diluted with EtOAc andwashed with water and brine (sat). The organic phase is dried overMgSO₄, filtered and then concentrated in vacuo.

Example 7:6-(bis((2-tert-butyl-sulfonylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol

2-tert-Butyl sulphonyl ferrocene carboxaldehyde (1 eq) is dissolved indry THF and treated with 6-aminohexan-1-ol (0.5 eq) and sodiumtrisacetoxyborohydride (2.5 eq) successively. The solution is allowed tostir at room temperature overnight. After this time the reaction isquenched by addition of NaHCO₃(sat). The organic layer is separated,then the aqueous layer back extracted with EtOAc. Combined organicextracts are dried over MgSO₄, filtered then concentrated in vacuo.

Example 8:6-(bis((2-tert-butyl-sulfidylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol

6-(bis((2-tert-butyl-sulfidylferrocenyl)1-methylferrocenyl)amino)hexan-1-olwas synthesised as shown in the scheme below.

1-[(Dimethylamino)methyl]-2-(t-butylthio)-ferrocene (1.21 g, 3.49 mmol)was dissolved in acetic anhydride (10 cm³). The brown solution was thenrefluxed for 1 hour, TLC at this indicated full consumption of thestarting material. The solution was allowed to cool to room temperature,the solution was then concentrated in vacuo to approximately 90% oforiginal volume. The resulting brown oil was then taken up in EtOAc (25cm³) and washed with NaHCO₃ (sat) (20 cm³) and brine (sat) (20 cm³). Thebrown solution was then dried over MgSO₄, filtered and concentrated invacuo to give the desired acetoxy ester as a orange/brown oil (1.12 g,93%) without need for further purification.

¹H NMR (250 MHz, C₆D₆) δ 5.37 (2H, d, J=1.43 Hz), 4.51 (1H, dd, J=2.6,1.4), 4.44 (1H, dd, J=2.6, 1.4), 4.07 (1H, t, J=2.6), 4.07 (5H, s) 1.82(s, 3H), 1.33 (9H, s).

To a suspension of lithium aluminium hydride (369 mg, 9.71 mmol) in Et₂O(15 cm³) at 0° C. was added the acetoxy ester (1.12 g, 3.23 mmol)dropwise via syringe. Once addition was complete the slurry was allowedto warm to room temperature and stir for 30 mins. After this time theflask was cooled to 0° C. and then quenched by sequential addition ofH₂O (369 μl), followed by 15% NaOH (aq) (369 μl) and H₂O (1.1 cm³) thesuspension was then allowed to warm to room temperature stirred for 10minutes, filtered and concentrated in vacuo to the desired product as anorange solid (790 mg, 80%) without the need for further purification.

¹H NMR (250 MHz, C₆D₆) δ 4.64 (2H, s), 4.41 (1H, dd, J=2.4, 1.5 Hz),4.32 (1H, dd, J=2.4, 1.5 Hz), 4.17 (5H, s), 4.08 (1H, t, J=2.6 Hz), 1.28(9H, s).

1-tert-butyl sulfidyl-2-hydroxymethyl ferrocene (304 mg, 1 mmol) andbarium manganate (1.02 g, 4 mmol) was placed in a schelnk tube, theflask sealed, then evacuated and back filled with argon four times andfinally left over an Argon atmosphere. The flask was then charged withbenzene (15 cm³) and the slurry was allowed to stir at room temperatureovernight. The slurry was then filtered through celite, and washed withEt₂O until the washing ran clear. The orange solution was concentratedin vacuo to give the desired product as a red oil (239 mg, 79%) withoutthe need for further purification.

¹H NMR (250 MHz, C₆D₆) δ 10.69 (1H, s), 5.08 (1H, dd, J=2.5, 1.3 Hz),4.47 (1H, dd, J=2.5, 1.3 Hz), 4.27 (1H, t, J=2.5 Hz), 4.13 (5H, s), 1.17(9H, s).

The 2-tert-butylsulfidyl-ferrocene carboxaldehyde (237 mg, 0.78 mmol)was placed in a round bottomed flask with 6-amino-hexanol (46 mg, 0.39mmol) and dissolved in dry THF (5 cm³).

The suspension was then treated with sodium trisacetoxyborohydride (322mg, 1.96 mmol). The flask was equipped with a condenser and refluxedovernight. After this time the flask was allowed to cool to roomtemperature. The reaction was quenched by addition of NaHCO₃(sat) (10cm³). Organics were separated and the aqueous layer back extracted withEtOAc (3×5 cm³). Combined organics were washed with brine (sat) (10cm³), dried over MgSO₄, filtered and concentrated in vacuo to give abrown oil. Purification by silica chromatography eluting with 50%EtOAc:nHex+2% TEA gave the desired product as an orange oil (18 mg, 7%)

¹H NMR (250 MHz, C₆D₆) δ 4.52 (2H, app s), 4.19-4.15 (14H, m), 3.91 (4H,s), 3.54 (2H, t, J=7.3), 2.64 (2H, t, J=7.3), 1.69-1.43 (26H, m); m/z(ESI μTOF, M+H) calcd for C₃₆H₅₂NOS₂Fe₂ m/z 690.2143 found 690.2158.Electrochemical potential (DPV) 369 mV.

Example 9:6-(bis((2-tert-butyl-sulfinylferrocenyl)-methylferrocenyl)amino)hexan-1-ol

6-(bis((2-tert-butyl-sulfinylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol was synthesised as shown in thescheme below.

3-tert-Butylsulfinyl ferrocene carboxaldehyde (26.5 mg, 0.083 mmol) wasdissolved in dry THF (1 cm³), treated with 6-amino-hexan-1-ol (4.8 mg,0.041 mmol). The brown solution was stirred for 10 mins before sodiumtrixacetoxyborohydride (34 mg, 0.2 mmol) was added in one portion. Thebrown suspension was then stirrer at room temperature overnight. Afterthis time the material was concentrated in vacuo to give a brown solid.Purifcation by silica chromatography eluting with 2.5% MeOH:CH₂Cl₂+2%NH₃OH to give the desired product as a yellow oil (4 mg, 14%)

¹H NMR (250 MHz, C₆D₆) δ 5.12 (2H, app s), 4.92 (2H, app s), 4.39 (14H,m), 4.22 (4H, s), 3.69 (2H, t, J=7.1 Hz), 2.45 (2H, t, J=7.1 Hz),1.50-1.18 (26H, m), m/z (ESI μTOF, M+H) calcd for C₃₆H₅₂NO₃S₂Fe₂ m/z722.2087 found 722.2089.m/z; Electrochemical potential (DPV) 532 mV.

Example 10: General Synthetic Procedure for Attaching PhosphoramiditeFunctional Group

The ferrocenyl derivative shown as a starting material in the abovereaction scheme is illustrative, and may be replaced by a molarequivalent of any of the compounds made in Examples 1 to 9 above.

N,N-diisopropylethylamine (0.4 mL, 8.4 mmol) was added to a stirredsolution of the ferrocene derivative (2.1 mmol) in dry THF (25 mL) undera nitrogen atmosphere. 2-cyanoethyldiisopropylchlorophosphoramidite (0.2ml, 3.15 mmol) was added dropwise and the resulting mixture was stirredfor 15 mins. MilliQ filtered water (200 mL) was added and the solutionwas stirred for a further 30 mins. Ethyl Acetate-Triethylamine (1:1, 25mL) was added, a precipitate formed. The mixture was washed withsaturated NaCHCO₃ (25 mL) and MilliQ filtered water (25 mL). The organicfraction was dried over MgSO₄ and the solvent was removed under vacuo.The crude product was then purified by silica gel chromatography(petroleum ether:ethyl acetate 9:1).

Example 11—Binding of Labels to Protein

The labels of the invention are attached to a peptide by attachment ofthe label to a free amine of, for example, a lysine residue in thepeptide. Attachment may be accomplished conventional techniquesincluding functionalisation of the labelling compound to form an activeNHS ester and reaction of the functionalised ester with the free aminegroup of the peptide.

Example 12—Binding of Labels to Microparticles

A biotin molecule is coupled to a label, for example a label as made inany of Examples 1 to 9. The biotinylation can be carried out in anautomated oligonucleotide synthesiser or using standard laboratoryconditions by reaction of ferrocenyl phosphoramidite label withN-hydroxysuccinimide (NHS) esters of biotin.

Paramagnetic treptavidin particles are washed ×3 (phosphate buffer) andmixed with biotinylated label, followed by incubation for 1 hour at roomtemperature with mixing. The particles are washed ×2 (phosphate buffer)and washed ×1 (PCR buffer). They are resuspended in final buffer (PCRbuffer). Following each wash step the supernatants are tested forelectrochemical signal, and if necessary washing is repeated until thesupernatants show no indication of free electrochemical label.

These particles are assayed at a range of concentrations to validatethat the observed electrochemical signal is attributable to the labelcoupled to the magnetic particles, using magnetic capture of theparticles and resuspension in a range of buffer volumes.

In Table 5 below, the electrode potentials of the compounds made inExamples 1 to 5, 8 and 9 are listed, together with the comparison valuefor N,N-diferrocenylmethyl-6-aminohexanol, a compound in which theferrocenyl groups are unsubstituted. A method for synthesis ofN,N-diferrocenylmethyl-6-aminohexanol is disclosed in WO2012/085591.

TABLE 5 Effect on electrode potential of substituents on ferrocenylmoieties Example Fc substituent Electrode potential A None 275 mV 11′-Iodo 442 mV 2 1′-(4-Nitrophenyl) 437 mV 3 1′-Furanyl 339 mV 42-di-t-butylphosphinyl 512 mV 5 1′-Chloro 452 mV 8 2-tert-butyl-sulfidyl369 mV 9 2-tert-butyl-sulfidyl 532 mV

The data in the above table shows that the compounds of Examples 1 to 5,8 and 9 provide useful electrochemically active labels. The labels maybe used to provide an electrochemical signal within a desired range ofvalues. They may be useful as alternative labels to other labellingcompounds with similar potential values, for example, where those otherlabelling compounds have disadvantageous properties in the assay inquestion, for example, incompatibility with impurities or othercomponents present in the assay or incompatibility with the measurementconditions, any of which could affect measurement sensitivity. As well,or instead, they may be used with one or more other labels in amultiplex assay in which more than one label is present to provide twoor more determinations in a single sample, the use of two or more labelswith different electrochemical properties in those circumstancespermitting effective distinction between measurements relating to therespective species to be determined.

1-54. (canceled)
 55. A method of detecting a nucleic acid, the methodcomprising: contacting the nucleic acid with a complementary nucleicacid probe under conditions to allow hybridization between said probeand an amplicon; and selectively degrading the probe, wherein the probeis labelled with a compound selected from the group consisting of L6-(bis((2-tert-butyl-sulfonylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol,6-(bis((2-di-tert-butyl-phosphinyl-ferrocenyl)1-methylferrocenyl)amino)hexan-1-ol,6-(bis((2-tert-butyl-sulfinylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol,an6-(bis((2-tert-butyl-sulfidylferrocenyl)1-methylferrocenyl)amino)hexan-1-ol.56. The method of claim 55, further comprising measuring electrochemicalactivity of the compound labelling the probe, wherein saidelectrochemical activity is dependent either quantitatively orqualitatively on the degradation of the probe.